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Biology of Reproduction 60, 499-507 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Relaxin Secretion and Gene Expression in Porcine Granulosa and Theca Cells Are Stimulated during In Vitro Luteinization1

Kathleen M. Ohleth3,a, and Carol A. Bagnell2,a

a Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08901-8525


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During formation of the corpus luteum, the primary source of relaxin switches from theca cells (TC) to granulosa-derived, large luteal cells. What controls this shift in relaxin production is poorly understood. The objective of this study was to observe the effect of luteinization on relaxin gene expression and secretion by porcine granulosa (GC) and TC using an in vitro model. TC and GC from medium-sized porcine follicles (4–6 mm) were treated for up to 8 days with LH (250 ng/ml) and/or insulin-like growth factor-I (IGF-I; 10 ng/ml). Media were assayed for relaxin and progesterone by RIA, changes in cell morphology were recorded, and total RNA was subjected to reverse transciption-polymerase chain reaction to monitor relaxin gene expression. In vitro luteinization, induced with LH + IGF-I treatment, was confirmed in both GC and TC by a change in morphology and a sustained, significant rise in progesterone secretion. In luteinizing GC, relaxin secretion was first detected after 5 treatment days, and steadily rose until it became significantly higher (p < 0.001) by treatment Days 7–8. In contrast, relaxin release from luteinizing TC was significant after only 2 days of treatment (p < 0.05) and increased consistently over the 8-day culture period (p < 0.001). In GC, relaxin mRNA was not detected until treatment Day 4 and became significantly higher (p < 0.001) by Day 8, the final treatment day. Relaxin transcript in luteinizing TC was low on treatment Days 2–4 and significantly higher (p < 0.01) by treatment Days 6 and 8. In summary, the present study demonstrates that hormones important in the control of luteinization are essential for regulating relaxin gene expression and secretion by GC and TC in the porcine follicle.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ovarian follicle has gained recognition as a source of relaxin in the pig. Relaxin has been detected in media from follicle wall cultures, ovarian extracts from prepubertal gilts, and follicular fluid from prepubertal, cyclic and pregnant animals [1–3]. The presence of relaxin mRNA and protein in theca cells (TC) from pig follicles established the TC as the main site of relaxin production in the porcine follicle [4–6]. However, as follicular cells differentiate to form the corpus luteum (CL), granulosa cells (GC) acquire the ability to produce relaxin [5, 7, 8]. What controls this switch in the cellular source of relaxin from the theca to the granulosa-derived luteal cell is unknown. However, several lines of evidence suggest that hormones controlling luteinization, such as LH, are important in regulating ovarian relaxin production. In vitro studies demonstrate relaxin production by luteinizing GC and point to gonadotropins as key modulators of relaxin secretion. Porcine GC from preovulatory follicles and luteinized human GC secrete relaxin in response to LH and hCG, respectively [9, 10]. In addition, administration of hCG raises plasma concentrations of relaxin in humans, baboons, and monkeys [11–13]. Furthermore, LH stimulates relaxin production from thecal explants of gonadotropin-primed, prepubertal gilts [14].

In CL of hysterectomized gilts, LH increases relaxin secretion in vivo and in vitro [15, 16], and insulin-like growth factor-I (IGF-I) alone or with LH augments in vitro secretion of relaxin [17]. The fact that IGF-I enhances LH-induced relaxin secretion is not surprising since IGF-I is widely accepted as an amplifier of gonadotropin action in the ovary [18, 19]. For example, IGF-I enhances gonadotropin-stimulated steroidogenesis from porcine TC and GC [20, 21]. Furthermore, IGF-I synergizes with LH to induce luteinization of porcine TC in vitro [22]. Similarly, IGF-I may contribute to any gonadotropin-induced regulation of relaxin gene expression and secretion that may occur during luteinization.

The first objective of the present study was to monitor relaxin gene expression and secretion during in vitro luteinization of porcine TC and GC. In vitro studies allow for the study of relaxin production in isolated follicular cell populations during differentiation. The second objective of this study was to determine whether hormones involved in luteinization, such as LH and IGF-I, could individually influence relaxin gene expression and secretion by porcine TC and GC in culture.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Porcine LH (pLH-B-I; AFP-5400) was donated by the National Hormone and Pituitary Program, NIDDK (Baltimore, MD). IGF-I was purchased from BACHEM Bioscience, Inc. (King of Prussia, PA). Collagenase (type II) and DNase I were obtained from Sigma Chemical Co. (St. Louis, MO). Fetal calf serum, certified (FCS), and all other tissue culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). [125I]Na for relaxin iodination was obtained from NEN Research Products, Inc. (Boston, MA). Monotyrosylated relaxin was a gift of Dr. R.V. Anthony (Colorado State University, Fort Collins, CO), and rabbit anti-porcine relaxin antibody (P5) was a gift of Dr. D.G. Porter (University of Guelph, Guelph, ON). The GeneAmp PCR Reagent Kit, including AmpliTaq polymerase, was purchased from The Perkin-Elmer Corp. (Norwalk, CT). Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and RNasin RNase inhibitor were obtained from Promega Corp. (Madison, WI), and random primers from Life Technologies, Inc. Polymerase chain reaction (PCR) primers for relaxin were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). PCR primers and competimers (alternate pair) for 18S ribosomal RNA (18S) were purchased from Ambion, Inc. (Austin, TX). The relaxin cDNA used to synthesize the relaxin cRNA probe was provided by Dr. S. Kwok (Albert Einstein Medical Center, Philadelphia, PA). The Gemini transcription system was purchased from Promega Corp. to synthesize the relaxin riboprobe.

Theca and Granulosa Cell Isolation and Culture

Ovaries from immature pigs (73–82 kg) were collected from a local slaughterhouse. Theca interna layers from medium-sized follicles (4–6 mm) were peeled away from the theca externa layer with fine forceps, and TC were harvested as described by May et al. [23]. GC were collected as thecal layers were deposited into a Petri dish containing medium 199 supplemented with 25 mM Hepes, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, and 0.5 µg/ml fungizone (M199). A modification of the method described by Kataoka et al. [24] was used to obtain the final theca cell preparations. Thecal layers were washed with M199 over a 177-µm mesh to remove loosely adhering GC. Remaining GC were washed away after the thecal layers were incubated with collagenase (0.05 mg/ml)/DNase I (1.25 µg/ml) for 10 min at 37°C. Thecal layers were then digested in collagenase (2 mg/ml)/DNase I (10 µg/ml) for 45 min at 37°C with intermittent Pasteur pipetting to obtain dispersed TC. Both GC and TC preparations were washed twice with M199. Viable TC (4 x 106 cells/well; 4.3 x 105 cells/cm2) and GC (6 x 106 cells/well; 6.4 x 105 cells/cm2) were seeded in 6-well culture dishes and incubated in a humidified chamber in an atmosphere of 5% CO2:95% air at 37°C. To facilitate attachment, TC and GC were initially cultured in M199 containing 10% FCS for 1 and 2 days, respectively. The 1-day attachment period for the TC eliminates GC contamination, since 2 days are required for GC to attach [23]. In addition, this method of TC isolation results in less than 3% contamination with GC [24].

To induce luteinization, TC and GC were treated up to 8 days with LH (250 ng/ml) and IGF-I (10 ng/ml) in the presence of 1% FCS [22]. Media were collected daily; and on Days 2, 4, 6, and 8 for GC and Days 2, 3, 4, 6, and 8 for TC, cell number was assessed in order to correct hormone secretion on a per-cell basis before RNA was isolated. In other experiments, TC and GC were incubated with 1% FCS in the presence or absence of LH (250 ng/ml) or IGF-I (10 ng/ml). In these experiments with individual treatments, TC and GC media were collected daily, and RNA was isolated on Days 2, 6, and 8 for GC, and Days 2, 4, and 6 for TC. The days of treatment are indicated by the letter "d" followed by the numerical day of treatment (i.e., Day 2 of treatment = d2).

RIAs

Progesterone (P4) was measured in the media to confirm luteinization of the TC and GC cultures. Media were appropriately diluted so that P4 could be measured directly using the Coat-A-Count P4 assay (DPC, Los Angeles, CA) according to the manufacturer's directions. Intra- and interassay variations were 7.9% and 9.9%, respectively, and assay sensitivity was 50.3 pg/ml. To monitor relaxin secretion by TC and GC, relaxin was measured in the media by RIA according to Afele et al. [25] with modifications. Briefly, monotyrosylated relaxin was iodinated with Iodogen according to the procedure of Hall et al. [26]. Samples of media (2.5 ml) were dried down with medium heat using an Integrated Speed-Vac System (Savant Instruments, Inc., Farmingdale, NY) and serially diluted in 0.05 M barbitone buffer containing 0.5% BSA. Anti-porcine relaxin antibody was incubated with the samples for 24 h at 4°C, and [125I]relaxin (12 000 cpm) was added for an additional 24 h at 4°C. Bound and free hormone were separated using conventional double-antibody procedures. Intra- and interassay variations were 7.6% and 10.5%, respectively, and assay sensitivity was 42 pg/ml. P4 and relaxin concentrations were corrected to cell number at the end of the incubation, since preliminary experiments showed that treatment with LH + IGF-I increased proliferation of GC and TC.

Isolation of Total RNA

Total RNA was isolated from the cultured cells according to the method of Peppel and Baglioni [27] with modifications. Since RNA and media for RIA were taken from the same cultures, cell number was assessed before RNA was isolated. Cells were harvested from the plates with 0.25% trypsin-1 mM EDTA, and cell number was determined from a small aliquot of cells by hemocytometer counting. Solution 1 (200 mM Tris HCl, pH 7.5; 1 mM EDTA; 2% SDS) was added to the remaining cells, and incubated for 10 min at room temperature. Solution 2 (150 µl; 4.4 M potassium acetate; 11.2% acetic acid) was then added and incubated on ice for 2 min. Precipitated DNA and proteins were pelleted by centrifugation at 14 000 rpm for 5 min at 4°C, and the supernatant was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and with chloroform:isoamyl alcohol (24:1). Total RNA was precipitated with one volume of ice-cold isopropanol for at least 1 h at -80°C and centrifuged at 14 000 rpm for 30 min at 4°C. The RNA pellets were washed with 80% ethanol, placed at -20°C for 10 min, centrifuged at 14 000 rpm for 20 min at 4°C, air-dried, dissolved in diethyl pyrocarbonate-treated water, and stored at -80°C. Quantity and purity of the RNA was assessed spectrophotometrically at 260 and 280 nm.

Reverse Transcription (RT)-PCR

Relaxin mRNA expression in cultured GC and TC was monitored using semi-quantitative RT-PCR [3]. Briefly, 1 µg total RNA was incubated at 37°C for 30 min in single-strength MMLV-RT reaction buffer; 1.5 µg random primers; 125 µM of deoxy (d) ATP, dCTP, dGTP, and dTTP; 1 U deoxyribonuclease (DNase) (ribonuclease [RNase]-free); and 20 U RNasin (RNase inhibitor). The DNase was inactivated for 7 min at 75°C, and 200 U MMLV-RT was added for a 45-min reaction at 42°C. A blank was included with each set of DNase-treated, RT reactions, in which sterile water was substituted for RNA. Subsequent PCR reactions were also performed without RT to confirm that DNA contamination was eliminated.

The primer sequences used to amplify porcine relaxin cDNAs were adopted from Knox et al. [28], and are specific for the 5'-(TACAGCAGCTGCAGTATCTA-OH) and 3'-(TGTCACTGAGAATACATGTG-OH) untranslated regions of the relaxin gene. Relaxin and 18S alternate primers:competimers (Ambion, Inc.) generated fragments of 663 and 324 base pairs (bp), respectively. Competimers for 18S amplification, chemically modified 18S primers, compete with the 18S primers and reduce the efficiency of the 18S amplification so that the 18S primers do not become limited. Five- and one-microliter aliquots of the RT reaction were used to amplify relaxin and 18S fragments, respectively, with the GeneAmp DNA Reagent Kit in the following 50-µl reaction: single-strength PCR buffer II; 2 mM MgCl2; 50 µM each of dATP, dCTP, dGTP, and dTTP; 10 pmol 5'- and 3'-relaxin primers or 18S primers:competimers (8:2; 4 µl); and 1.25 U AmpliTaq polymerase. Amplification of relaxin and 18S cDNAs was performed for 30 and 15 cycles, respectively, each cycle consisting of 1-min denaturation at 95°C, 45-sec annealing at 56°C, and 1-min extension at 72°C in the Ericomp DeltaCycler I System (Ericomp, Inc., San Diego, CA). Each set of PCR reactions included the appropriate volume of the RT blank. A pool of follicle RNA from pigs 60 and 72 h after treatment with eCG, which contains relaxin mRNA [6], and liver from neonatal pigs were used as positive and negative controls for relaxin transcript, respectively [3]. The post-eCG follicle pool was included in each set of RT-PCR reactions to account for variability between each set of reactions. To verify that relaxin and 18S cDNAs were amplified in the exponential range, an RT reaction of the post-eCG follicle pool was serially diluted, and each dilution was used as a template for relaxin and 18S PCR reactions [3].

Southern Analysis

Relaxin antisense riboprobe was generated from an EcoRI and SphI digest of a relaxin cDNA (pPR308–6) in pUC18, corresponding to an 797-bp region of the reported porcine relaxin cDNA sequence [29]. The cDNA fragment was ligated into the pGEM-4Z vector, linearized with SphI, and transcribed with SP6 polymerase to synthesize an antisense relaxin cRNA probe. Southern hybridization was performed as previously described [3]. Briefly, PCR products were fractionated on 5% polyacrylamide-Tris-borate-EDTA, stained with ethidium bromide, and photographed. Gels containing relaxin PCR fragments were prepared for Southern analysis by denaturing and neutralizing before electrophoretic transfer to nylon membranes. Nucleic acids were fixed and UV-cross-linked to the membrane. Relaxin antisense riboprobe was labeled with biotin by irradiation with long-wave UV light in the presence of psoralen-biotin. The biotin-labeled relaxin probe was then used to detect relaxin PCR products. The membranes were prehybridized at 42°C for 15 min and then hybridized with 10 ng/ml biotinylated relaxin riboprobe in hybridization buffer overnight at 42°C. After posthybridization washes, relaxin antisense probe hybridized to relaxin PCR products was detected using the BrightStar BioDetect Nonisotopic Detection Kit (Ambion, Inc.). The membranes were washed, blocked, and incubated with a streptavidin-alkaline phosphatase conjugate for 30 min. After a series of washes, membranes were incubated in the CDP-Star chemiluminescent reagent for 5 min. After 24 h, the membranes were exposed to x-ray film, and PCR product bands were scanned to obtain optical densities. To correct for variability between individual Southern blots, the positive control, post-eCG follicle PCR products from the appropriate set of RT-PCR reactions, were run on each gel. For each blot or gel, optical densities generated from the relaxin and 18S PCR products of each sample were divided by the densitometric values of the relaxin and 18S cDNA fragments from the positive control sample. Subsequently, optical densities of the relaxin cDNAs were corrected to the optical density of the 18S cDNAs of the same sample.

Statistics

Results are presented as the mean ± SE of at least three experiments using independent pools of GC and TC. Data were analyzed by ANOVA, and comparisons were made with Duncan's multiple-range test. Values of p < 0.05 were accepted as significant. Relaxin and P4 secretion on different treatment days were compared within each treatment. In the Results section, hormone secretion and gene expression values are compared with respect to values before treatment, unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphological Changes in Porcine GC and TC

Control GC appeared fibroblastic during the entire treatment period (Fig. 1a). By the second day of treatment with LH and IGF-I, GC exhibited a distinct change in morphology from fibroblastic to epithelioid in shape, characteristic of morphological luteinization, and this shape persisted throughout the treatment period (Fig. 1b; d4 shown). This morphological transformation was accompanied by an accumulation of lipid droplets and granules in the cytoplasm and an increase in the cytoplasmic/nuclear ratio. Alterations in GC morphology in response to LH treatment were less pronounced than those of cells treated with LH and IGF-I combined, in that not all cells underwent morphological transformation (data not shown). The appearance of GC treated with IGF-I alone was similar to that of control cells (data not shown). Control TC (Fig. 1c) remained fibroblastic throughout the 8 days of treatment. After d3-d4 of LH and IGF-I treatment, small clusters of morphologically luteinized TC were dispersed among the fibroblastic cells (Fig. 1d). In response to IGF-I alone, TC also formed clusters of epithelioid-like cells (data not shown). However, the IGF-I-induced TC clusters were fewer in number and contained fewer cells than TC treated with both LH and IGF-I. TC incubated with LH alone did not exhibit any morphological changes when compared to controls (data not shown).



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  		FIG. 1. GC and TC morphology in response to LH + IGF-I. After attachment, GC and TC were incubated with LH (250 ng/ml) and IGF-I (10 ng/ml). Cell morphology was photographed at 2-day intervals up to 8 days. Morphology of cells treated after 4 days of treatment is shown. Arrows point to clusters of morphologically luteinized TC. a) GC control (CON). Original magnification x68 (reproduced at 96%). b) GC treated with LH + IGF-I. Original magnification x68 (reproduced at 96%). c) TC control (CON). Original magnification x34 (reproduced at 96%). d) TC treated with LH + IGF-I. Original magnification x34 (reproduced at 96%).

P4 and Relaxin Secretion by Porcine GC and TC in Response to LH + IGF-I

TC and GC treated with LH and IGF-I to induce luteinization (Figs. 2 and 3) secreted significantly higher levels of P4 than did control TC and GC (Fig. 4) throughout the treatment period (p < 0.001). Since P4 release from control TC and GC did not differ significantly throughout the 8-day treatment period, P4 secretion by each cell type on different days of luteinization treatment was compared. The sustained increase in P4 secretion by TC and GC treated with LH and IGF-I confirmed in vitro luteinization of both cell types (Figs. 2 and 3). Therefore, GC and TC treated with LH and IGF-I are referred to as luteinizing GC and TC. Luteinizing GC secreted significantly higher amounts of P4 by d2 than did GC before hormone treatment (p < 0.01; Fig. 2). After 2 days of LH and IGF-I exposure, GC P4 secretion remained elevated from d2 to d6, then significantly rose on d7 and d8 (p < 0.001; Fig. 2). P4 release from luteinizing TC was significantly enhanced after 2 days of hormone exposure when compared to P4 in TC media before treatment (p < 0.01; Fig. 3). On subsequent days, TC P4 secretion rose steadily, and it peaked on d4 and d5 (p < 0.001; Fig. 3). By d7, there was a precipitous decline in P4 release from TC when compared to that on d5 (p < 0.001; Fig. 3). At the end of the 8-day treatment period, P4 in TC media was similar to that before treatment (Fig. 3). The amount of P4 in GC-conditioned media over the 8-day treatment period was approximately 5-fold higher than that of the TC-conditioned media.



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  		FIG. 2. Relaxin and P4 secretion from luteinizing GC. After attachment, GC were treated with LH (250 ng/ml) and IGF-I (10 ng/ml) with 1% FCS up to 8 days (n = 3). Media were collected daily and assayed for relaxin and P4 by RIA. In media of control GC (1% FCS; not shown), relaxin was not detected; P4 did not vary over the 8-day period and averaged 38.3 ± 5.1 ng/106 cells/24 h. Different superscripts denote significant differences (p < 0.05) in hormone secretion on different treatment days.



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  		FIG. 3. Relaxin and P4 secretion from luteinizing TC. After attachment, TC were treated with LH (250 ng/ml) and IGF-I (10 ng/ml) up to 8 days (n = 3). Media were collected daily, and relaxin and P4 were measured by RIA. In media of control TC (1% FCS; not shown), relaxin was not detected; P4 did not vary over the 8-day period and averaged 19.4 ± 8.8 ng/106 cells/24 h. Different superscripts denote significant differences (p < 0.05) in hormone secretion on different treatment days.



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  		FIG. 4. P4 secretion from GC and TC in response to hormones. After attachment, GC and TC were treated with LH (250 ng/ml) or IGF-I (10 ng/ml) up to 8 days (n = 3). Media were collected daily, and P4 was measured by RIA. *Significant differences (p < 0.05) in hormone secretion on different treatment days within each hormone treatment. Note the order-of-magnitude difference in the y-axis for P4 secretion compared to the same y-axis in Figures 2 and 3.

Luteinizing TC and GC secreted increasing amounts of relaxin as the treatment period progressed (Figs. 2 and 3). Since there was no evidence for relaxin in the media of control TC and GC, relaxin concentrations in the media of luteinizing TC and GC were compared over the treatment period. In GC, relaxin was first detected in media on d5, and secretion significantly increased by d7 and d8 (p < 0.01; Fig. 2). In contrast, relaxin in media from TC increased as early as d2 (p < 0.05) and continued to rise through d7 (p < 0.001; Fig. 3). By d8, there was a slight decline in relaxin secretion by TC relative to d7 (p < 0.05; Fig. 3). Overall, the pattern of relaxin secretion paralleled, but lagged behind, that of P4 secretion in both GC and TC.

P4 and Relaxin Secretion by Porcine GC and TC in Response to LH or IGF-I

When TC and GC were treated with LH or IGF-I, both cell types secreted significantly less P4 than did TC and GC treated with LH and IGF-I combined (p < 0.001). For example, d2 exposure of GC to LH + IGF-I resulted in over 4 times more P4 secretion (Fig. 2; 9122 ng/106 cells/24 h) than that from GC incubated with LH alone for 2 days (Fig. 4; 2139 ng/106 cells/24 h). Secretion of P4 from control and IGF-I-treated GC and TC did not vary over the 8-day treatment period (Fig. 4). However, after d1 of LH treatment, GC released significantly higher amounts of P4 than did the control GC, and secretion remained elevated until d3 (p < 0.001; Fig. 4). On d4 of LH treatment, GC P4 secretion remained significantly higher than that of control P4 secretion (p < 0.01; Fig. 4), although it had declined with respect to d3 of treatment (p < 0.001). By d5, P4 release in LH-treated GC was similar to that of controls and GC before treatment (Fig. 4). Whereas TC incubated with LH exhibited a peak of P4 secretion on d1 (p < 0.001; Fig. 4), P4 levels in media from LH-treated TC dropped by d2 and remained similar to those of controls until the end of the treatment period (Fig. 4). Relaxin was not detected in TC and GC-conditioned media of cells treated with LH or IGF-I alone.

Relaxin Gene Expression in Porcine GC and TC

The relaxin and 18S cDNAs were amplified in the exponential range as a linear relationship existed between PCR product yield and the serially diluted RT reaction used as the PCR template ([3]; data not shown). There was no evidence of relaxin mRNA in control GC or, by d2, in GC treated with LH and IGF-I (Fig. 5a). Relaxin gene expression was detected in luteinizing GC by d4 (Fig. 5, a and b) and became significantly higher by d8 than on d2 (p < 0.01; Fig. 5, a and b). Relaxin mRNA was observed at low levels in control TC (data not shown), and in luteinizing TC on d2 to d4 (Fig. 6a). By d6 and d8, relaxin transcript in TC was significantly enhanced by LH and IGF-I treatment when compared to d2 to 4 of treatment (p < 0.001; Fig. 6, a and b). Relaxin gene expression was not detected in GC treated with LH or IGF-I alone. In TC, low levels of relaxin mRNA did not increase in response to LH or IGF-I treatment on d2-d4. However, by d6, IGF-I-treated TC expressed significantly higher relaxin transcript than did control and LH-treated TC (p < 0.01; data not shown). Relaxin mRNA was not detected in the negative control pig liver sample ([3]; data not shown).



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  		FIG. 5. Relaxin gene expression in luteinizing GC. GC were treated as described in Figure 2. Total RNA was collected on d2, d4, d6, and d8. RT-PCR followed by nonradioactive Southern hybridization was used to detect relaxin mRNA in each sample. Differences in relaxin transcript between samples were calculated as described in the Materials and Methods. a) Relaxin and 18S fragments from a representative pool of control and treated GC. b) Densitometric values from all treated GC cultures (n = 3). Different superscripts denote significant differences (p < 0.05) in relaxin gene expression on different treatment days. +, Positive control: post-hCG porcine follicle pool.



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  		FIG. 6. Relaxin gene expression in luteinizing TC. TC were treated as described in Figure 3. Total RNA was collected on d2, d3, d4, d6, and d8. RT-PCR followed by nonradioactive Southern hybridization was used to detect relaxin mRNA in each sample. Differences in relaxin transcript between samples were calculated as described in the Materials and Methods. a) Relaxin and 18S fragments from a representative pool of treated TC. b) Densitometric values from all treated TC cultures (n = 3). Different superscripts denote significant differences (p < 0.05) in relaxin gene expression on different treatment days. + Positive control: post-hCG porcine follicle pool.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report is the first to demonstrate a direct effect of in vitro luteinization on relaxin gene expression and secretion by TC and GC of the porcine ovary. When porcine GC and TC were treated with LH and IGF-I to induce in vitro luteinization, thecal relaxin gene expression and secretion was enhanced, and de novo synthesis of relaxin from porcine GC was induced. In contrast, treatment of GC and TC with LH or IGF-I alone did not stimulate detectable levels of relaxin production from GC or TC. The extent of relaxin gene expression and secretion found in luteinizing GC and TC appeared to mirror the magnitude of luteinization, as characterized by marked morphological changes and sustained P4 secretion.

The hallmarks of GC luteinization include an increase in P4 secretion with a concurrent change in cell morphology [30–32]. The combination of LH and IGF-I in the presence of serum, used to promote luteinization, elicited the most distinct changes in morphology of porcine TC and GC, from fibroblastic to epithelioid in shape. LH-induced changes in morphology were less striking than the morphological changes seen with LH and IGF-I combined. For example, not all GC became epithelioid as observed in GC treated to induce luteinization. The more pronounced morphological changes in GC resulting from treatment with a combination of hormones are consistent with previous reports. When Maruo et al. [33] incubated porcine GC with FSH, IGF-I, or FSH + IGF-I, cells treated with FSH + IGF-I became epithelioid, while cells treated with FSH or IGF-I alone retained a fibroblastic appearance. Along the same lines, full morphological luteinization of porcine GC was not achieved when cells were incubated with LH and FSH until insulin, cortisol, and thyroxine were added to the cultures [34].

In the present study, the amount of P4 secreted was consistent with morphological changes. When cells were incubated with LH and IGF-I, a greater amount of P4 in both GC- and TC-conditioned media was observed than when cells were treated with LH or IGF-I alone. Our results are consistent with previous studies demonstrating that IGF-I enhances gonadotropin-induced steroidogenesis. For example, in porcine, bovine, and ovine GC, IGF-I augments FSH-stimulated P4 release [20, 35–37]. IGF-I also stimulated gonadotropin-induced P4 secretion from porcine and bovine TC [21,38]. In other studies, insulin, which is reported to interact with the IGF-I receptor [20, 33, 39], synergistically increased FSH- and hCG-stimulated P4 secretion from porcine and bovine GC [40–42], and LH-stimulated P4 production by porcine and bovine TC [38, 43]. Although LH alone significantly elevated P4 secretion from both GC and TC, this enhanced P4 release was not sustained. A similar trend in P4 secretion was observed by Channing [30, 31] when porcine GC from 3- to 5-mm follicles were incubated with LH over time. In these studies, although LH significantly stimulated P4 production from GC when compared to control GC, LH-stimulated P4 release significantly decreased with treatment over time [30,31]. Additionally, Lino et al. [44] found that insulin was required for LH- and FSH-stimulated P4 secretion by porcine GC after 5 days in culture. Thus, the data presented here support earlier studies in which a combination of hormones is required to maintain elevated P4 secretion when compared to individual hormones. Collectively, the present morphological and steroidogenic data indicate that a combination of gonadotropins and growth factors is most effective at stimulating GC and TC function.

The present data also demonstrate that IGF-I did not significantly influence secretion of P4 from TC and GC. In agreement with the present study, IGF-I alone did not enhance P4 production by bovine TC [38, 45]. In addition, while IGF-I increased total P4 content in porcine GC [39] and P4 production by bovine GC [44], Baranao and Hammond [20] observed that IGF-I induced P4 production by porcine GC only in the presence of FSH. This inconsistency between reports may be dependent on a variety of factors including differences in culture conditions and hormone concentrations. For instance, P4 secretion from porcine TC [21] and bovine GC [36] was stimulated by IGF-I at 50 and 200 ng/ml, respectively, doses relatively high compared to the 10 ng/ml IGF-I used in the present study. Veldhuis et al. [39, 46] reported that 10 ng/ml IGF-I stimulated a small but significant rise in total P4 content from porcine GC. However, the use of 1% FCS in the present study may have masked any significant effect of IGF-I on P4 release. Likewise, Channing et al. [34] found that P4 secretion was lower from GC in serum-containing media than in serum-free cultures. The follicular source of GC may also influence the variation in IGF-I effects on P4 production in the different studies. In ovine GC, IGF-I dose-dependently enhanced P4 secretion by GC from large follicles; however, IGF-I had no effect on GC from small follicles [35]. While Veldhuis et al. [39, 46] reported effects of IGF-I on steroidogenesis in porcine GC pooled from small and medium follicles, little information is available on the effects of IGF-I on porcine GC from medium follicles alone.

During formation of the CL, histological evidence suggests that TC and GC of the follicle differentiate into small and large luteal cells, respectively [45, 47, 48]. At this time in the pig, the primary source of relaxin shifts from the TC of the follicle to the GC-derived large luteal cells [5, 7, 8]. The results from our in vitro experiments, the first to demonstrate de novo relaxin synthesis in porcine GC, are in agreement with in vivo studies in which neither relaxin transcript nor protein was detected in GC-derived large luteal cells until Day 9 of the cycle [7, 8]. Similarly, in our cultures, when GC were treated with LH and IGF-I, relaxin mRNA and secretion were not observed until d4 and d5, respectively. While in vivo reports show that theca-derived luteal cells of the immature CL express relaxin protein between Days 5 and 7 of the cycle and relaxin gene from 54 h to 9 days after ovulation, relaxin is not produced in the theca-derived luteal cell of the mature CL [7, 8]. Therefore, the enhanced thecal relaxin gene expression and secretion during in vitro luteinization reported here is in agreement with in vivo thecal relaxin production during early, but not late, CL development. The reason for this contrast between the in vitro and in vivo studies is unclear. One possibility is that the time frame of the present in vitro studies may be more representative of early, rather than late, CL development. Alternatively, this high relaxin mRNA in luteinizing TC in vitro may be due to the absence of some factor from luteinizing GC that may act in a paracrine manner to suppress thecal relaxin gene expression in vivo, later in CL formation. In summary, these results indicate that GC acquire the ability to express relaxin as they differentiate into luteal cells, and that luteinization is important in the regulation of relaxin gene expression and secretion in vitro.

In the present study, relaxin secretion and gene expression were not detected from GC or TC treated with LH alone. In contrast, other studies have reported that LH enhanced relaxin secretion in vitro from preovulatory porcine GC [9], luteinized human GC [10], and thecal explants from gonadotropin-primed pigs [14]. In these past studies, factors other than in vitro gonadotropin exposure may have had an impact on relaxin release. The follicular tissue used in these previous studies was collected when systemic gonadotropins were high, which most likely created in vivo follicle conditions quite different from those of the present in vitro studies. For example, elevated gonadotropins can create locally high growth factor concentrations in the follicle [49], which may have contributed to LH-stimulated relaxin release in the previous studies. In our studies, GC and TC were collected from medium-sized follicles of immature animals. Therefore, our GC and TC cultures were not pre-exposed to preovulatory levels of gonadotropins or the local hormonal milieu resulting from these gonadotropin levels. This lack of in vivo exposure to gonadotropins may explain the absence of a detectable amount of relaxin in the media of follicular cells treated with LH alone. By definition, luteal cells have been exposed to gonadotropins during differentiation, which may be a requirement for IGF-I to enhance follicular relaxin secretion. This idea is supported by studies in CL from hysterectomized gilts, in which IGF-I stimulates in vitro basal and LH-stimulated relaxin secretion [16].

The data reported here support the idea that control of P4 and relaxin secretion are linked, since although relaxin secretion lagged behind P4 release, the overall pattern of P4 secretion paralleled that of relaxin in both TC and GC. A similar scenario in relaxin and P4 secretion is reported in human granulosa lutein cultures treated with hCG [50]. However, several in vivo reports demonstrate that P4 is not required for relaxin production. While the P4 inhibitor, trilostane, decreased P4 levels in rhesus monkeys during the menstrual cycle and simulated pregnancy, plasma relaxin concentrations remained similar to those in animals administered vehicle [51, 52]. Additionally, in the utero-ovarian vein of periparturient sows, relaxin content peaked 1–2 days before birth, when P4 levels were falling or had already declined [53]. Further investigation is required to determine which factor(s), in addition to the gonadotropins and growth factors controlling luteinization, may be important for granulosa-lutein relaxin synthesis. In a study in rats, Goldsmith et al. [54] found that upon hysterectomy on d16 of pregnancy, serum relaxin promptly fell, suggesting that a placental and/or fetal factor is important in maintaining luteal relaxin production.

The present study traced relaxin gene expression and secretion during in vitro luteinization of porcine GC and TC. Luteinization induced by a combination of LH and IGF-I was required for the up-regulation of relaxin gene expression and secretion by GC and TC. Conversely, LH or IGF-I alone were ineffective in modulating relaxin mRNA and release. Collectively, our results demonstrate that the combination of LH and IGF-I is most effective in stimulating differentiation and relaxin production in porcine GC and TC. In addition, the results presented here were similar to those of in vivo studies monitoring relaxin synthesis in GC and TC during formation of the CL. In conclusion, these studies indicate that hormones important in the control of luteinization are essential in the regulation of relaxin production by follicular cells of the pig ovary. Further studies are required to determine the physiological role of relaxin in the pig follicle at the time of ovulation and early CL development.


   ACKNOWLEDGMENTS
 
The authors wish to acknowledge the assistance of Judy Lenhart, Kimberly Kirkup, Stacey Santoro, and undergraduate students in our laboratory; Dr. R.V. Anthony for monotyrosylated relaxin; Dr. D.G. Porter for the relaxin antisera; Dr. S. Kwok for the relaxin cDNA; the National Hormone and Pituitary Program NIDDK for porcine LH; Dr. S.V. Radecki for statistical assistance; and Leidy's, Inc. for the supply of pig ovaries.


   FOOTNOTES
 
1 Supported by USDA #93–37203–8979 and New Jersey Agricultural Experiment Station #D-06125–2-98 (to C.A.B.). Back

2 Correspondence: Carol A. Bagnell, Department of Animal Sciences, Rutgers University, 84 Lipman Drive, New Brunswick, NJ 08901–8525. FAX: 732 932 6996; bagnell{at}aesop.rutgers.edu Back

3 Current address: Kathleen M. Ohleth, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033. Back

Accepted: September 29, 1998.

Received: July 31, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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